The recent Ebola virus (EBOV) outbreak in Western Africa, which introduced the virus to non-African nations including the United States, highlights the imminent threat to global health posed by filoviruses and the urgent need for basic and translational efforts. A critical step in the viral lifecycle of filoviruses is the replication of the non-segmented, negative strand RNA genome by the viral RNA dependent RNA polymerase (RDRP) complex. The EBOV RDRP complex is composed of the large protein (L), nucleoprotein (NP), viral protein 35 (VP35) and viral protein 30 (VP30) and is essential for transcription of viral mRNA and replication of antigenomic and genomic RNA. During RNA synthesis, components of the RDRP complex undergo multiple structural rearrangements to make the NP-associated RNA template accessible to the L polymerase while protecting the NP-RNA template from cellular nucleases. Despite the importance of the EBOV RDRP complex to pathogenesis and the potential as therapeutic targets, insights into RDRP complex assembly and its regulatory interactions with host or viral proteins are lacking. We will address these longstanding mechanistic questions in this PPG. Project 2 will use biochemical and hybrid structural methods that will combine results from NMR, X-ray crystallography, small angle X-ray scattering (SAXS) and cryo-electron microscopy (cryoEM) in order to define the molecular mechanism of the EBOV RDRP complex assembly and function, and to define its regulatory mechanisms and structural dynamics. Our strong preliminary results support a working model where conformational remodeling of the RDRP complex is controlled by cis- and trans-acting factors. We have assembled a highly productive and collaborative team of investigators with complementary expertise to perform the following Aims: 1. Define the molecular mechanism by which EBOV VP35 facilitates delivery of NP in the RNA-free form. 2. Elucidate the internal dynamics of the NP protein and define the role of different NP conformations in viral RNA synthesis. 3. Determine the molecular architecture of the EBOV RDRP complex by hybrid structural methods. 4. Determine the structural basis for cis-acting RNA elements identified in Project 1 and novel host factors from Project 3/Core B that regulate EBOV RDRP complex. Completion of these studies will address longstanding and critical mechanistic questions in EBOV biology that are also central to a better understanding of non-segmented negative sense RNA viruses (NNSVs). These insights are also expected to yield new opportunities for antiviral development.